This invention is in the field of cell biology. Specifically, the invention relates to the generation of a cell array comprising a multiplicity of cell types. Such array can be used to generate multiple test units containing cells of identical type and passage. The compositions and methods embodied in the present invention are particularly useful for rapid identification of differential gene expression patterns and protein-protein interaction patterns, as well as for high throughput screening of candidate modulators of signal transduction pathways.
The imminent completion of sequences of the entire human genome will provide a wealth of information on gene sequences, and genome structure and organization. The acquisition of the genome sequences of multiple model organisms will further open up new avenues to search for the biological significance of these data. The next objective is to harness this vast wealth of genetic data in the prediction, diagnosis and treatment of diseases. In particular, methods are required which will allow one to distinguish differential gene expression patterns between cells of different organisms, between different cell types of the same organism, or between different pathological stages of the same cell. Additional techniques are needed for recordation and correlation of the temporal changes in cell physiology in response to a variety of external stimuli. Methods of this type are denoted xe2x80x9cfunctional genomicsxe2x80x9d, which aims at delineating the relationship between the phenotype of a cell with its genotype at any given time.
Delineating the genotypic characteristics contributing to the phenotypic traits of a given cell type has until now been a daunting task. Traditional approaches for identifying genes or gene products unique to a particular type of cell are generally highly limited, targeting at only one, or a few specific gene sequences, and analyzing one cell type at a time. This is primarily due to the fact that maintaining multiple cell lines or types of cell cultures is extremely costly and labor intensive. Recently developed techniques such as micro-patterned arrays (described in WO 97/45730, WO 98/38490) and microfluidic arrays provide valuable tools for comparative cell-based analysis, but they also have pronounced limitations. To the extent that these techniques employ living cells whose characteristics may not remain constant from one experiment to another, inherent variability associated with cells carried in different facilities or with varying passages is inevitably being introducing during experimentation. It is a well-known problem in the art that both genotypic and phenotypic characters of cells may change over time when cultured in vitro.
There thus remains a considerable need for devices and methods of performing comparative cell-based analyses with minimum inconsistencies. An ideal device would allow (a) the cellular activities of multiple types of cells to be examined simultaneously; (b) the same batches of cells of multiple types to be tested during multiple rounds of experiments, so as to minimize the variability in cell conditions; and finally, the devices must support high throughput screening for candidate therapeutic targets and/or agents in a cost effective fashion. The present invention satisfies these needs and provides related advantages as well.
A principal aspect of the present invention is the design of a technique capable of generating multiple copies of a miniaturized cell array comprising a variety of cell types. This technique of cell-array production simplifies the laborious and expensive procedures of culturing multiple types of cells each time when needed. This technique allows multiple rounds of biological assays, or assays carried out at different facilities in different geographical locations, to be conducted on cells having essentially the same characteristics as those used in a previous experiment, and thus minimizes experimental variations in cell conditions often encountered when dealing with cells of different batches, varying passages, and of different laboratory or depository origin.
Accordingly, the present invention provides a method of preparing a cell array that comprises the following steps: (a) providing an array of tubes, each tube having at least one lumen and a population of cells that is contained within said lumen; (b) cross-sectioning the array of tubes to yield a plurality of transverse tube segments; and (c) immobilizing the plurality of tube segments on a solid support.
The present invention also provides a tube having a maximum length in the range of about 0.01 micron to about 5 mm. The tube has at least one lumen and a population of cells that is contained and immobilized within the lumen. In one aspect of this embodiment, the tube has a transverse sectional area of about 0.01 mm2 to about 5 cm2. In another aspect, the tube is made of one or more substances selected from the group consisting of plastic polymer, glass, cellulose, nitrocellulose, semi-conducting materials, and metal. In yet another aspect, the tube contains a population of cells that is embedded in a matrix. The matrix can be made of one or more of the substances selected from the group consisting of methocellulose, laminin, fibronectin, collagen, agar, Matrix-gel(copyright), OCT compound, and paraffin.
In a separate aspect of this embodiment, the population of cells contained in the tube is substantially homogenous. The cells can be living or dead cells; eukaryotic or prokaryotic cells; embryonic or adult cells; or cells of ectodermal, endodermal or mesodermal origin. The cells loaded in the tube can also be freshly isolated cells, cultured cells in either primary or secondary cultures, or cells of an established cell line. Furthermore, the cells may be wildtype, genetically altered or chemically treated cells.
The present invention further provides a cell array comprising a plurality of the tubes embodied in the invention. In one aspect of this embodiment, each tube of the cell array is immobilized on a solid support. The solid support on which tubes of cells are arrayed can be flexible or rigid. Preferably, the solid support is made of plastic polymer, glass, cellulose, nitrocellulose, semi-conducting material, metal, or any combination thereof. A preferred cell array comprises at least two tubes having an exposed upper transverse sectional surface. Optionally, polynucleotides contained in the tubes of cells are denatured.
In another aspect, at least a subset of the tubes in the cell array comprises cells of a unique type. The tubes in the subset may have multiple lumens, wherein each lumen of the tube within the subset contains a cell population that is unique with respect to all other cell populations contained in other lumens of the tubes of the subset. In an alternative, the tubes in the subset may have multiple lumens, wherein each lumen of the tube within the subset contains a cell population that is unique with respect to all other cell populations contained in other lumens of the same tube. Each tube of a cell array may contain at least 10 cells of the same type, preferably 100 cells of the same type. The cell array may optionally contain tubes of control cells.
In a separate aspect of this embodiment, the cells contained in the tubes of the subset of tubes differ in one or more of the characteristics selected from the group consisting of genotypic characteristics, species origin, developmental stage, developmental origin, tissue origin, cell-cycle point, chemical treatment and disease state. Whereas the species origin may be selected from the group consisting of human, mouse, rat, fruit fly, worm, yeast and bacterium, suitable tissues from which cells are derived are blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids. The cells contained in the subset of tubes of the array may also differ in developmental stage including embryo and adult stages, as well as developmental origin such as ecotodermal, mesodermal, and ectodermal origin. As such, the invention cell arrays encompass embryonic cell arrays, adult cell arrays, primary cell arrays, cell line arrays, tissue arrays, mammalian cell arrays, zoo arrays, personal cell arrays, genetically altered cell arrays, chemically treated cell arrays, and disease cell arrays. A preferred disease cell array is a cancer cell array.
Also provided in the present invention are methods of using the above described cell arrays. In one embodiment, the present invention provides a method of simultaneously detecting the presence of a specific protein-protein interaction involving a proteinaceous probe and a target protein in multiple types of cells. The method involves the steps of: (a) providing a subject cell array; (b) contacting a proteinaceous probe that is specific for a target protein with the array of tubes under conditions sufficient to produce a stable probe-target complex; and (c) detecting the formation of the stable probe-target complex in each tube, thereby detecting the presence of specific protein-protein interaction in multiple types of cells. Examples of proteinaceous probes that may be employed in the assay are antibodies, cell surface receptors, secreted proteins, receptor ligands, immunoliposomes, immunotoxins, cytosolic proteins, nuclear proteins, and functional motifs thereof. Examples of target proteins that may be detected are membrane proteins, secreted proteins, cytosolic proteins, nuclear proteins and chaperon proteins. In certain aspects, the target protein is differentially expressed in one or more cell types contained in the array of tubes.
In another embodiment, the present invention provides a method of determining cell-type binding selectivity of an antibody using the cell arrays.
In yet another embodiment, the present invention provides a method of detecting differential expression of a target protein in a multiplicity of cell types derived from at least two subjects. Such method involves: (a) staining a first cell array of claim 16 with an antibody that is specific for the target protein, wherein the array comprises a plurality of tubes containing a multiplicity of cell types of a first subject; (b) detecting the stain in each tube of the array that forms a first immunostaining pattern representative of the differential expression of said target in the multiple types of cells of the first subject; (c) staining a second cell array of claim 16 with an antibody that is specific for the target protein, wherein the array comprises a plurality of tubes containing a multiplicity of cell types of a second subject; (d) detecting the stain in each tube of the second array that forms a second immunostaining pattern representative of the differential expression of said target in the multiple types of cells of the second subject; and (e) comparing the immunostaining patterns, thereby detecting the differential expression of the target protein in the multiplicity of cell types of the subjects.
In yet another embodiment, the invention provides a method of detecting differential representation of a target polynucleotide in a multiplicity of cell types.
In yet another embodiment, the invention provides a method of detecting differential representation of a target polynucleotide in a multiplicity of cell types derived from at least two subjects.
In yet still another embodiment, the invention includes a method for identifying a modulator of a signal transduction pathway. Such method comprises the steps of (a) providing a cell array as described above, wherein at least a subset of the tubes on the array contains cells expressing at least one reporter molecule that yields a detectable signal transduction readout; (b) contacting the array with a candidate modulator; and (c) assaying for a change in the signal transduction readout, thereby identifying a modulator of the signal transduction pathway.
In addition, the invention encompasses computer-implemented methods for detecting differential expression of a target polynucleotide or protein in a multiplicity of cell types. Also included are computer-based systems for detecting differential expression of a target polynucleotide or protein in a multiplicity of cell types derived from at least two subjects. Further provided by the present invention are kits for simultaneously detecting the presence of a target polynucleotide or polypeptide in a multiplicity of cell types comprising the subject cell arrays in suitable packaging.